The elucidation of molecular alterations that occur during human breast cancer development may permit the identification of preventative strategies for women at high risk. Lesions such as atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS) confer 4- and 8-10 fold increases in risk for the development of invasive carcinoma, respectively. We examined a cohort of approximately 100 human biopsy specimens using in situ hybridization to examine trends in mRNA expression levels, with the goal of identifying genes whose expression patterns correlated with increasing risk. We have reported that cyclin D mRNA levels were quantitatively elevated in a majority of DCIS lesions, but not in the lower risk ADH or other benign lesions. Current studies are examining the potential functional effects of cyclin D overexpression using transfection of human MCF-10A and derivative breast cell lines. After using five different transfection strategies on this very difficult system, we have successfully transfected the TG1 and NeoT sublines, and will conduct in vitro and in vivo analysis of cell cycle, proliferation, histopathology, tumorigenicity, DNA repair, etc. We have also asked if preventative regimens currently under study can elevate breast cell cyclin D expression. We have found that retinoids elevate cyclin D expression in vitro, with a concomitant increase Rb phosphorylation and G1/S cell cycle progression. Similar in situ hybridization experiments have been conducted with the RXR form of the retinoic acid receptor. We found that only 8% of benign lesions, but 66% of noncomedo DCIS and 88% of comedo DCIS lesions overexpress RXR mRNA. To investigate this trend further at the protein level, we have developed immunohistochemistry for the alpha, beta and gamma forms of RXR using antigen retrieval. RXR-alpha is the predominant form expressed in breast epithelium and myoepithelium; biopsy samples are currently being evaluated for protein expression trends. These data have identified cyclin D and the RXR retinoic acid receptor as genes whose expression levels increase in biopsy specimens with increased risk for the development of invasive breast carcinoma. These genetic events can be hypothesized to be functionally relevant in the pathogenesis of breast cancer, testable by transfection experiments.